Detection of Carbamazepine and Its Metabolites in Blood Samples by LC-MS/MS / 法医学杂志

OBJECTIVES@#To establish a method for the detection of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS).@*METHODS@#The blood samples were treated with 1-butyl-3-methylimidazolium hexafluorophosphate as an extraction solvent. The samples were extracted by ultrasound-assisted extraction and separated by ZORBAX Eclipse Plus C18, 95Å column. The mobile phase A aqueous solution containing 0.1% formic acid and 10 mmol/L ammonium acetate, and mobile phase B mixed organic solvent containing acetonitrile/methanol (Vacetonitrile∶Vmethanol=2∶3) were used for gradient elution at the flow rate of 1.00 mL/min. An electrospray ion source in positive mode was used for detection in the multiple reaction monitoring.@*RESULTS@#The linearities of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples were good within the corresponding range, with correlation coefficients (r) greater than 0.995 6. The limits of detection were 3.00, 0.40 and 1.30 ng/mL, respectively. The limit of quantitation were 8.00, 1.00 and 5.00 ng/mL, respectively. The extraction recoveries ranged from 76.00% to 106.44%. The relative standard deviations of the intra-day and inter-day precisions were less than 16%. Carbamazepine and its main metabolite 10,11-dihydro-10,11-epoxycarbamazepine were detected in blood samples of death cases with a mass concentration of 2.71 μg/mL and 252.14 ng/mL, respectively.@*CONCLUSIONS@#This method has high sensitivity and good selectivity, which is suitable for the detection of carbamazepine and its metabolites in blood samples, and can be used for carbamazepine-related forensic identifications.

Regulatory effect of eight Chinese herbal medicines on glucose and lipid metabolism and their potential active components based on HCBP6 target / 中国中药杂志

With the improvement of living standards and changes in working style, the prevalence of abnormal glucose and lipid metabolism in humans is increasing in modern society. Clinically, the related indicators are often improved by changing the lifestyle and/or taking hypoglycemic and lipid-lowering drugs, but there are no therapeutic drugs for disorders of glucose and lipid metabolism at present. Hepatitis C virus core protein binding protein 6(HCBP6) is a newly discovered target that can regulate triglyceride and cholesterol content according to level oscillations in the body, thereby regulating abnormal glucose and lipid metabolism. Relevant studies have shown that ginsenoside Rh_2 can significantly up-regulate the expression of HCBP6, but there are few studies on the effect of Chinese herbal medicines on HCBP6. Moreover, the three-dimensional structural information of HCBP6 has not been determined and the discovery of potential active components acting on HCBP6 is not rapidly advanced. Therefore, the total saponins of eight Chinese herbal medicines commonly used to regulate abnormal glucose and lipid metabolism were selected as the research objects to observe their effect on the expression of HCBP6. Then, the three-dimensional structure of HCBP6 was predicted, followed by molecular docking with saponins in eight Chinese herbal medicines to quickly find potential active components. The results showed that all total saponins tended to up-regulate HCBP6 mRNA and protein expression, where gypenosides showed the optimum effect on up-regulating HCBP6 mRNA and ginsenosides showed the optimum effect on up-regulating HCBP6 protein expression. Reliable protein structures were obtained after the prediction of protein structures using the Robetta website and the evaluation of the predicted structures by SAVES. The saponins from the website and literature were also collected and docked with the predicted protein, and the saponin components were found to have good binding activity to the HCBP6 protein. The results of the study are expected to provide ideas and methods for the discovery of new drugs from Chinese herbal medicines to regulate glucose and lipid metabolism.

Optimization of determination of astragaloside Ⅳ in Astragali Radix by continuous single-factor method / 中国中药杂志

This paper aims to solve the problems of complicated-unstable test solution preparation process and insufficient extraction of the active ingredient astragaloside Ⅳ in the legal method for the determination of astragaloside Ⅳ in Astragali Radix. The continuous single-factor analysis of seven main factors affecting the content of astragaloside Ⅳ was carried out by HPLC-ELSD, and then the pre-paration method of test solution was optimized. This optimized method exhibited excellent performance in precision, repeatability and stability. The average recovery rate of astragaloside Ⅳ was 99.65% with RSD 2.2%. Astragaloside Ⅳ showed a good linearity between the logarithm of peak area and the logarithm of injection quantity in the range of 0.46-9.1 μg(r=0.999 6). The contents of astragaloside Ⅳ in 29 batches of Astragali Radix were determined by the new and the legal methods. The results showed that the average content of astragaloside Ⅳ in these Astragali Radix samples determined by the former method was 1.458 times than that of the latter one, indicating the new method was simple, reliable and more adequate to extract target compound. According to the results, it is suggested to improve the content standard of astragaloside Ⅳ in Astragali Radix in the new edition of Chinese Pharmacopeia.

Determination of β-nicotinamide mononucleotide and nicotinamide adenine dinucleotide in Dendrobium officinale and congeneric species by UPLC-MS/MS / 中国中药杂志

As anti-aging ingredients, β-nicotinamide mononucleotide(NMN) and nicotinamide adenine dinucleotide(NAD~+) have attracted worldwide attention in recent years. After oral administration, NMN can be converted into NAD~+ in vivo and the latter is the actual ingredient which exerts anti-aging effect. In order to explore the "rejuvenating and anti-aging" effect of Dendrobium officinale, which was firstly recorded in Shennong's Herbal Classic of Materia Medica, this study established the quantitative method of UPLC-MS/MS for simultaneous determination of NMN and NAD~+ in D. officinale and the congeneric species for the first time, and 34 batches of samples were detected. UPLC conditions are as follows: ACQUITY UPLC HSS T3 column(2.1 mm × 100 mm, 1.8 μm), gradient elution with acetonitrile-0.1% formic acid in water at the flow rate of 0.3 mL·min~(-1), and column temperature of 40 ℃. MS conditions were scanned electrospray ionization source and multiple reaction monitoring mode. The method was verified by systematic methodology. The mean recoveries of NMN and NAD~+ were 77.58% and 80.70%, respectively, with RSD of 3.6% and 4.3%, separately. All results showed that the content of NMN was higher in D. officinale than in the other congeneric species. Particularly, the content in fresh D. officinale stems was as high as 0.931 9 μg·g~(-1). NAD~+ was only found in D. officinale and the content was three times higher than that of NMN. This may be the reason that D. officinale topped the "nine famous anti-aging herbs". In addition, processing method influences the content of NMN and NAD~+ in Dendrobium. Specifically, the content of NMN and NAD~+ was in the order of fresh Dendrobium stems > dried Dendrobium stem segments > spiral or spring-like dried Dendrobium stems.

Rapid discovery of tyrosinase inhibitors from Sophora flavescens:significance for comprehensive utilization of its non-medicinal resources / 中国中药杂志

Sophorae Flavescentis Radix,derived from the root of Sophora flavescens in the Leguminosae family,has been widely used in the medicine,agriculture,animal husbandry,and daily chemical industry. A pharmacophore model-based method for rapid discovery of tyrosinase inhibitors( TIs) from S. flavescens was established by molecular docking under Lipinski rules,and verified by enzyme assays. Briefly,the chemical constituent database of S. flavescens( CDSF) was established based on the previous papers. Theoptimal pharmacophore model( OPM) was constructed by DS 2019 on the basis of known active TIs. Eighty-three hits predominated by flavonoids having higher fitting scores with OPM than the positive control were screened out,and subjected to molecular docking based on the three-dimensional structure of tyrosinase crystal protein. The potential TIs such as kurarinone and nor-kurarinone were rapidly discovered from the compounds with higher docking scores than the positive control under the Lipinski rules. The results were verified by the in vitro enzyme assays. The inhibition activities of tyrosinase from non-medicinal parts of S. flavescens were also tested to explore the relationship between the inhibition activity and chemical compositions. This study is expected to provide data support for the comprehensive application and development of S. flavescens and also a new method for the rapid discovery of active substances or functional constituents in the complex systems.

Scientificity of traditional commodity grade of Chinese medicinal materials--taking Lonicerae Japonicae Flos as example / 中国中药杂志

Taking Lonicerae Japonicae Flos as an example, the method of "expert consensus of different regions" was used to screen the representative samples and evaluate their commodity grades. The correlation analysis, hierarchical cluster analysis and partial least squares discriminant analysis(PLS-DA) of "commodity grade-appearance characteristic-component content" were carried out to reveal the scientificity of traditional commodity grade of Chinese medicinal material. By referring to the existing literature and the grade investigation from the sample collection regions, 78 "initial grade" samples were screened out from 118 collected samples. Authoritative experts from four regions(n=4) including Linyi(Shangdong province), Bozhou(Anhui province), Anguo(Hebei province) and Beijing were organized to evaluate their commodity grades, separately. Based on the grade consistency rate(R_i≥70%), 69 "local grade" samples were screened out from the "initial grade" samples. Based on the average grade consistency rate ■ "authoritative grade" samples were screened out from the "local grade" samples, including15 first-grade samples, 9 second-grade samples, 11 third-grade samples and 17 fourth-grade samples. For these "authoritative grade" samples, the main appea-rance characteristics were quantified and the contents of 13 components were determined by ultra performance liquid chromatography(UPLC). Furthermore, the total contents of 6 phenolic acids, 4 flavonoids and 3 iridoids were calculated, respectively. The results of correlation analysis showed that 4 appearance characteristics indices were correlated with the commodity grades: color, rate of yellow bars(including blooming flowers), rate of black heads(including black bars), and rate of stems and leaves(including bud debris). Five component content indices were correlated with the commodity grades: chlorogenic acid, isochlorogenic acid C, sweroside, loganin and the total contents of six phenolic acids. Furthermore, chlorogenic acid, loganin and the total contents of six phenolic acids showed significantly negative correlation with the main appearance characteristics, indicating that the appearance characteristics of Lonicerae Japonicae Flos can reflect its internal quality, and these 3 indices can be used as quality markers(Q-markers). The results of hierarchical cluster analysis showed that the samples of four grades were classified into four categories, and the samples with the same grades and the same categories accounted for 80.8% of the total samples, while the samples with the different grades were obviously classified into different categories. The results of PLS-DA analysis showed that the samples of different grades showed obvious intragroup aggregation and intergroup dispersion. The above results indicated that it was feasible to evaluate the traditional commodity grade of Lonicerae Japonicae Flos by the method of "expert consensus of different regions". For the evaluation of traditional commodity grade of Chinese medicinal material, the samples should be representative, expert conclusions should have enough consensuses, and grade determination should be authoritative. As the crystallization of clinical experience, traditional commodity grade can scientifically reflect the internal quality of Chinese medicinal material.

Synthesis and activity evaluation of allyl-quercetin derivatives / 中国药学杂志

OBJECTIVE: To design and synthesize two kinds of allyl-substituted quercetin and investigate their anti-oxidation and anti-tumor activities in vitro. METHODS: The target compounds 1 and 2 were first synthesized from quercetin by hydroxyl protection, allylation,Claisen rearrangement and deprotection. The anti-oxidation and anti-tumor activities of the target compounds and intermediate products were evaluated by DPPH and MTT assay. RESULTS: Eight compounds were synthesized,including six intermediates and two target compounds,in which four were new compounds. All of them were confirmed by 1H-NMR,13C-NMR and LC-MS spectra. The anti-oxidation and anti-tumor tests showed that compounds 1,2,5,6,8 and 9 had anti-oxidation activities and compound 5 inhibited A549 lung cancer cell proliferation. Compound 9 could inhibit the proliferation of lung cancer A549 cells and HepG2 cells. CONCLUSION: The compounds with electron-donating groups have significant anti-oxidant activity. When acetyl and methyl ether groups are used as the protecting groups,the position of introducing allyl group to quercetin has obvious impact on the anti-tumor activity.